I want to screen a Pseudomonas aeruginosa mutant which would have a lacZ gene after being conjugated with E.coli. But unfortunately, on xgal plates i endup having accumulation of blue color patches making screening a difficult process. Why does my plate turn to blue patches? I have also cross checked if Ecoli itself or pseudomonas itself are being major contributes for such colorations. But Ecoli, as expected dint grow on IPTG/BCIG/TET plates and the plates were all normal. Pseudomonas itself were not able to produce any betagactosidase activity as well. I end up having blue plates only after conjugating ecoli (with lacZ vector) and pseudomonas (Recipient) ? any suggestions ????
DR ASWIN!!
Why are some colonies blue and some white?
Any colony containing the plasmid (and therefore the functioning β-galactosidase gene) will turn blue, a result of the β-galactosidase activity. ... The insert disrupted the β-galactosidase gene, and therefore these colonies remain white.
For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. ... The desired recombinant colonies can be easily picked and cultured. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. TD-P Revision 3.0 ... Utilizing X-Gal and IPTG Plates ... To perform blue-white screening after transformation, X-Gal is added along with Isopropyl β-D-. 1-thiogalactopyranoside (IPTG), an inducer of lacZ ω gene expression. ... produce the blue color, because they do not produce functional β-galactosidase
https://www.goldbio.com/documents/1031/Blue%20White%20Screening%20of%20Bacterial%20Colonies%20using%20X-Gal%20and%20IPTG%20Plates.pdf
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